Human embryonic stem (hES) cells are pluripotent such that they can differentiate into all three germ layers, thus potentially all different types of tissues of the body. Pluripotency is characteristic of only embryonic cells, but it can also be achieved by reprogramming differentiated cells by transferring nuclear contents into unfertilized, enucleated oocytes or by fusing with ES cells. To achieve the initial embryo-like state, it is a pre-requisite to be able to maintain and propagate these ES cells in culture conditions in vitro. Currently, such recipe exists for mouse ES cells. Surprisingly, similar media components for hES cells do not work. This very first technical barrier needs to be overcome in order to realize full clinical potential of stem cell therapy. We propose to develop a novel recipe of chemically defined culture media and culture conditions to grow and maintain pluripotency of hES cells. The media we will evaluate are combinatorial mixtures containing only recombinant proteins, chemically synthesizable reagents, or human source factors. To achieve new sets of recombinant protein reagents known to be involved in controlling differentiation and pluripotency of embryo-like cells, we will develop a novel biochemical strategy of producing a set of target protein reagents effectively in test tubes. To screen conditions using these chemically defined components and various culture conditions, we will develop a new cell line containing a reporter gene (GFP) recombined into human Oct4 gene. Human Oct4 gene is the prominent marker for stemness of the hES cells. There are three specific Aims for this proposed study. They are, 1) production of the media components biochemically, 2) development of two Oct4-reporting hES cell lines, and 3) screening of culture media and conditions for maintaining pluripotency of hES cells. These experiments will be carried out in parallel as collaboration between two laboratories {REDACTED}. Once Aims 1 and 2 are completed, we will evaluate these hES cell lines in various culture conditions systematically (Aim 3). In doing these high-throughput assays for functional characterization, we will also conduct screening of known chemical library of selected drugs and metabolites to glean into their potential ability to augment or inhibit actions of the engineered biologic reagents in controlling the growth and pluripotency of hES cells. From the screening using these two cell lines, we will establish the firm method of propagating and maintaining pluripotency of hES cells for subsequent clinical applications.

Statement of Benefit to California:

Establishing the methods to promote and maintain pluripotency of human embryonic stem cells is a groundwork absolutely necessary to facilitate stem cell research in general and to be adopted for its immediate clinical applications. For instance, the proposed study will generate essential data to facilitate biotechnological approaches to scale up to meet the industrial demand of the much needed protein reagents to culture hES cells. These reagents we established are also critical for basic research of developmental and cell biology, structural biology, and drug discovery. So scientific and industrial benefits to California are enormous.

The proposed study requires combination of extensive biochemistry and developmental biology expertise. Development of new techniques and reagents for the maintenance and proliferation of pluripotent hES cells are fundamentally essential in order to fully exploit therapeutic potentials of hES cell therapy. We believe that the prime benefit from results of the proposed study is to add substantially to the body of knowledge on growing, maintaining, and finally guiding hES cells to the differentiated states as needed to develop effective therapeutic means.

Review Summary:

SYNOPSIS: In this proposal, which is highly responsive to the RFA, the goal is to improve culture conditions for hES cells by focusing on TGF-beta signaling components. Cells will be generated with a GFP cassette expressed under Oct4 control, and then cells will be screened for growth in defined media containing one or more of 22 different TGF-ligands that have been produced by recombinant methods. The "product" of this work will be culture conditions that maintain pluripotency of hES cells.

SIGNIFICANCE AND INNOVATION: Improved culture conditions that maintain pluripotency of hES cells are a worthy goal. This application is novel for the production of 22 recombinant TGF-ligands for screening in vitro. While not particularly innovative, this project has the potential to generate extremely useful information for the field as a whole. This is the sort of fundamental research that is needed for ultimate progress in using hES cells, but the approach is not necessarily one that would usually be feasible outside of industry.

STRENGTHS: The prior cloning and production of a large number of TGF-beta ligands is an important aspect of this proposal. The proposed screening, one that could yield a breakthrough in optimizing a defined culture condition that could become standard in the field, is straightforward and logical and thus likely to yield useful information. The project brings together outstanding expertise from a protein chemist/structural biologist (Choe) and a stem cell biologist (Belmonte). This concerted effort, rather than reliance on one or two components, is a strength of the proposal. Few groups could carry out such an ambitious “production factory” and there is even the possibility that novel factors could be engineered.

WEAKNESSES: A potential weakness may relate to the focus on TGF-beta components. Nonetheless, there is sufficient prior data from mES cells to recommend consideration of these factors. A more minor weakness is that Oct4:GFP lines are already generated, and it was not made clear why the proposed line will be better. Reviewers recommend the use of the BAC-Oct4 reporter rather than targeting one of the Oct4 loci by homologous recombination. The latter approach will inactivate one Oct4 allele, and this may be detrimental to maintenance of the pluripotent state.

DISCUSSION: This is a well thought out, well written, well focused proposal that very few people could execute; there is a huge number of combinations to test in this robotic screen to define optimal culture conditions. The approach is practical with straight-forward testing procedures, and the growth studies using 22 different ligands, already in-hand, is a real strength. Homologous recombination (HR) in hESC is an issue as reviewers questioned whether this group has the necessary experience with this technique since only 2 groups have reported success with HR in hES cells. A BAC-based approach may provide some information if the homologous integration approach fails. Information from this study is likely to affect the whole field.